Mycobiota dynamics and mycotoxin detection in PGI Salame Piemonte.

AIMS: The complex mycobiota that colonizes traditional fermented sausages plays an important role in the organoleptic properties of such products. The aim of the present study was to investigate fungal diversity and mycotoxin production during maturation of PGI Salame Piemonte. METHODS AND RESULTS: Casing and meat samples were collected at five sampling times from three different batches produced in the same factory and analysed using culture-dependent and independent approaches. Penicillium nalgiovense, which was deliberately inoculated, and Debaryomyces hansenii were the most dominant taxa in casings. Several other fungi mainly belonging to Penicillium crustosum, Penicillium glabrum, Penicillium nordicum, Cladosporium spp., Candida sake, Candida zeylanoides and Yarrowia divulgata were also identified. The casing mycobiota was compared to that of the meat using a metataxonomic approach and a higher fungal diversity was observed in meat as compared to casings. Mycotoxins and penicillin G were monitored using QTOF LC-MS and only trace amounts of roquefortine C were detected in two batches. CONCLUSIONS: The present study highlighted the diversity of Salame Piemonte mycobiota and the important contribution of autochthonous fungi to its diversity. The absence of mycotoxins and penicillin G confirmed the high hygienic quality of the studied product regarding fungal and mycotoxin contamination. SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time, this study provides insights about Salame Piemonte mycobiota, which together with the bacterial microbiota and Salame Piemonte process specifications, are responsible for the product organoleptic properties.

New insights into the haemo- and coelo-microbiota with antimicrobial activities from Echinodermata and Mollusca.

AIMS: The aim of this study was to investigate the diversity of bacteria with antimicrobial activity present in the coelomic fluid and haemolymph of wild and healthy echinodermata and mollusca. METHODS AND RESULTS: Collection expeditions of healthy marine molluscs and echinoderms were conducted in the Glenan archipelago in spring 2014. Members of the culturable microbiota present in the haemolymph, (haemo-microbiota) of Haliotis tuberculata (gastropoda, abalone) and Mytilus edulis (bivalvia, mussel), as well as in the coelomic fluid (coelo-microbiota) of Echinus esculentus (echinoidea, sea urchin) and Holothuria forskali (Holothuroidea, holothurian) were screened for antimicrobial activity, and further identified using 16S rRNA sequencing. Except for E. esculentus, culturable bacteria in the internal fluids of all studied organisms (mussel, abalone and holothurian) were more abundant than in seawater. The haemo- and coelo-microbiota with antimicrobial activity differed significantly between host species, in terms of abundance and diversity. Indeed, higher numbers were isolated from mussel than from abalone haemolymph. Moreover, in mussels and holothurians, bacteria with antimicrobial activities were predominantly Vibrio spp. (respectively 55 and 45%), while Pseudoalteromonas spp. were the most abundant (50%) in abalone haemolymph. Nevertheless, the activity spectra of these bacteria mainly included marine pathogens affiliated to the Vibrio genus. CONCLUSION: The haemo- and coelo-microbiota with antimicrobial activities were significantly related to their host species and differed in terms of abundance and diversity. These bacteria may play a key role in host homeostasis against pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: This study brings new knowledge on the diversity of bacteria present in the internal fluids of two marine molluscs and two echinoderms and their antimicrobial activities towards marine pathogens.

Implementation of an FTIR spectral library of 486 filamentous fungi strains for rapid identification of molds.

Filamentous fungi may cause food and feed spoilage and produce harmful metabolites to human and animal health such as mycotoxins. Identification of fungi using conventional phenotypic methods is time-consuming and molecular methods are still quite expensive and require specific laboratory skills. In the last two decades, it has been shown that Fourier transform infrared (FTIR) spectroscopy was an efficient tool for microorganism identification. The aims of this study were to use a simple protocol for the identification of filamentous fungi using FTIR spectroscopy coupled with a partial least squares discriminant analysis (PLS-DA), to implement a procedure to validate the obtained results, and to assess the transferability of the method and database. FTIR spectra of 486 strains (43 genera and 140 species) were recorded. An IR spectral database built with 288 strains was used to identify 105 different strains. It was found that 99.17% and 92.3% of spectra derived from these strains were correctly assigned at the genus and species levels, respectively. The establishment of a score and a threshold permitted to validate 80.79% of the results obtained. A standardization function (SF) was also implemented and tested on FTIR data from another instrument on a different site and permitted to increase the percentage of well predicted spectra for this set from 72.15% to 89.13%. This study confirms the good performance of high throughput FTIR spectroscopy for fungal identification using a spectral library of molds of industrial relevance.

Application of capillary electrophoresis single-stranded conformation polymorphism (CE-SSCP) analysis for identification of fungal communities in cheese.

As major contributors of the ripening process, yeasts and filamentous fungi play a fundamental role in cheese-making. Still, there is no rapid and affordable identification method available for both yeasts and filamentous fungi encountered in cheeses. In the present study, we developed a method based on CE-SSCP analysis of nuclear ribosomal DNA ITS amplicons, along with a species pattern database comprising 37 fungal species. By combining analyses of the ITS1 and ITS2 conformers, 25 out of 37 species were discriminated using CE-SSCP analysis. This reproducible and sensitive method was applied to determine the fungal community composition of 36 cheeses including blue-veined, pressed-cooked, pressed-uncooked, red-smear and surface-mould ripened cheeses. Overall, each cheese contained between 1 and 6 fungal species and 23 different species of fungi were detected including 8 yeast species, 9 filamentous species and 6 unidentified species. Comparison of the fungal diversity obtained after cloning and sequencing (rDNA ITS) versus CE-SSCP for 8 cheeses showed that CE-SSCP was at least as exhaustive as cloning and sequencing of thirty clones per cheese. In conclusion, this CE-SSCP method was an effective tool to identify the fungi present in various cheese varieties and may be of interest for the cheese industry to rapidly describe the composition of cheese fungal communities.

Dynamics of metabolically active bacterial communities involved in PAH and toxicity elimination from oil-contaminated sludge during anoxic/oxic oscillations.

The kinetics of polycyclic aromatic hydrocarbons (PAH) elimination from a contaminated sludge were determined in bioreactors under different conditions: continuously oxic, anoxic, and anoxic/oxic oscillations. The dynamics of metabolically active bacterial communities and their involvement in PAH degradation were followed by T-RFLP targeting 16S rRNA and ring hydroxylating dioxygenase (RHD) transcripts, respectively. PAH degradation was related to toxicity elimination using an aryl hydrocarbon receptor-responsive reporter cell line. Oxygen supply was identified as the main factor affecting the structure of bacterial communities and PAH removal. PAH-degrading bacterial communities were stable throughout the experiment in all conditions according to the presence of RHD transcripts, indicating that bacterial communities were well adapted to the presence of pollutants. Oxic and anoxic/oxic oscillating conditions showed similar levels of PAH removal at the end of the experiment despite several anoxic periods in oscillating conditions. These results highlight the role of dioxygenase activity after oxygen addition. Nevertheless, the higher toxicity elimination observed under oxic conditions suggests that some metabolites or other unidentified active compounds persisted under oscillating and anoxic conditions. Our results emphasize the importance of using complementary biological, chemical and toxicological approaches to implement efficient bioremediation strategies.

Application of denaturing high-performance liquid chromatography (DHPLC) for yeasts identification in red smear cheese surfaces.

AIMS: To evaluate and optimize the use of denaturing high-performance liquid chromatography (DHPLC) for yeasts identification in red smear cheese surfaces. METHODS AND RESULTS: The resolution of DHPLC was first evaluated and optimized using a mixture of PCR amplicons of the internal transcribed spacer 2 (ITS2) region of 19 yeast reference strains representing 18 species that are common in the cheese microbiota. Sixteen of the 18 yeast species could be resolved by combining runs at temperatures of 57.5 and 59 degrees C. Then, DHPLC was used to investigate the yeast microbiota of pasteurized Maroilles, Munster and Livarot cheese surfaces by comparing their peak profiles with our reference yeast database and by collecting/sequencing of peak fractions. Debaryomyces hansenii and Geotrichum candidum for Munster and Maroilles cheeses, and Candida catenulata, Candida intermedia and G. candidum for Livarot cheese were identified using the reference database and collecting/sequencing of peak fractions. CONCLUSIONS: DHPLC technique was found to have good resolution properties and to be useful for investigating the yeast microbiota of red smear cheese surfaces. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time that DHPLC is applied to study the yeast microbiota of red smear cheese surfaces.

Trafficking of Shigella lipopolysaccharide in polarized intestinal epithelial cells.

Bacterial lipopolysaccharide (LPS) at the apical surface of polarized intestinal epithelial cells was previously shown to be transported from the apical to the basolateral pole of the epithelium (Beatty, W.L., and P.J. Sansonetti. 1997. Infect. Immun. 65:4395-4404). The present study was designed to elucidate the transcytotic pathway of LPS and to characterize the endocytic compartments involved in this process. Confocal and electron microscopic analyses revealed that LPS internalized at the apical surface became rapidly distributed within endosomal compartments accessible to basolaterally internalized transferrin. This compartment largely excluded fluid-phase markers added at either pole. Access to the basolateral side of the epithelium subsequent to trafficking to basolateral endosomes occurred via exocytosis into the paracellular space beneath the intercellular tight junctions. LPS appeared to exploit other endocytic routes with much of the internalized LPS recycled to the original apical membrane. In addition, analysis of LPS in association with markers of the endocytic network revealed that some LPS was sent to late endosomal and lysosomal compartments.

Effects of Proteus vulgaris growth on the establishment of a cheese microbial community and on the production of volatile aroma compounds in a model cheese.

AIMS: To investigate the impact of Proteus vulgaris growth on a multispecies ecosystem and on volatile aroma compound production during cheese ripening. METHODS AND RESULTS: The microbial community dynamics and the production of volatile aroma compounds of a nine-species cheese ecosystem were compared with or without the presence of P. vulgaris in the initial inoculum. Proteus vulgaris was able to colonize the cheese surface and it was one of the dominant species, representing 37% of total isolates at the end of ripening with counts of 9.2 log(10) CFU g(-1). In the presence of P. vulgaris, counts of Arthrobacter arilaitensis, Brevibacterium aurantiacum and Hafnia alvei significantly decreased. Proteus vulgaris influenced the production of total volatile aroma compounds with branched-chain aldehydes and their corresponding alcohols being most abundant. CONCLUSIONS: Proteus vulgaris was able to successfully implant itself in a complex cheese ecosystem and significantly contributed to the organoleptic properties of cheese during ripening. This bacterium also interacted negatively with other bacteria in the ecosystem studied. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time that the impact of a Gram-negative bacterium on cheese microbial ecology and functionality has been described.

Stability of the biodiversity of the surface consortia of Gubbeen, a red-smear cheese.

A total of 1,052 bacteria and 828 yeasts were isolated from the surface flora of 6 batches of Gubbeen cheese made in 1996-1997 and 2002-2003. Stability of the microflora was evaluated over time and also during ripening at 4, 10, and 16 d (batches 4, 5, and 6) or at 4, 16, 23, and 37 d (batches 1, 2, and 3). Bacteria were identified using pulsed-field gel electrophoresis, repetitive extragenic palindromic-PCR, and 16S rRNA gene sequencing, and yeasts were identified by Fourier transform infrared spectroscopy. The bacteria included at least 17 species, of which the most common were Staphylococcus saprophyticus (316 isolates), Corynebacterium casei (248 isolates), Brevibacterium aurantiacum (187 isolates), Corynebacterium variabile (146 isolates), Microbacterium gubbeenense (55 isolates), Staphylococcus equorum/cohnii (31 isolates), and Psychrobacter spp. (26 isolates). The most common yeasts were Debaryomyces hansenii (624 isolates), Candida catenulata (135 isolates), and Candida lusitaniae (62 isolates). In all batches of cheese except batch 2, a progression of bacteria was observed, with staphylococci dominating the early stages of ripening and coryneforms the later stages. No progression of yeast was found. Pulsed-field gel electrophoresis showed that several different strains of the 5 important species of bacteria were present, but generally only one predominated. The commercial strains used for smearing the cheese were recovered, but only in very small numbers early in ripening. Four species, B. aurantiacum, C. casei, C. variabile, and Staph. saprophyticus, were found on all batches of cheese, but their relative importance varied considerably. The results imply that significant variation occurs in the surface microflora of cheese.

[Some comments on the biological properties of calcitonin: an eventual new therapeutic utilization (author's transl)]. / Quelques remarques sur les propriétés biologiques de la calcitonine: une utilisation thérapeutique nouvelle possible.

The calcitonin-adrenal antagonism brings the authors to propose a new therapeutic use of calcitonin. That hormone could minimize the effects of externe stimulations and its use as sedative is proposed.

Differentiation and identification of filamentous fungi by high-throughput FTIR spectroscopic analysis of mycelia.

Routine identification of fungi based on phenotypic and genotypic methods can be fastidious and time-consuming. In this context, there is a constant need for new approaches allowing the rapid identification of molds. Fourier-transform infrared (FTIR) spectroscopy appears as such an indicated method. The objective of this work was to evaluate the potential of FTIR spectroscopy for an early differentiation and identification of filamentous fungi. One hundred and thirty-one strains identified using DNA sequencing, were analyzed using FTIR spectroscopy of the mycelia obtained after a reduced culture time of 48 h compared to current conventional methods. Partial least square discriminant analysis was used as a chemometric method to analyze the spectral data and for identification of the fungal strains from the phylum to the species level. Calibration models were constructed using 106 strains pertaining to 14 different genera and 32 species and were used to identify 25 fungal strains in a blind manner. Identification levels of 98.97% and 98.77% achieved were correctly assigned to the genus and species levels respectively. FTIR spectroscopy with its high discriminating power and rapidity therefore shows strong promise for routine fungal identification. Upgrading of our database is ongoing to test the technique's robustness.

Biodiversity of antifungal lactic acid bacteria isolated from raw milk samples from cow, ewe and goat over one-year period.

Antifungal lactic acid bacteria (ALAB) biodiversity was evaluated in raw milk from ewe, cow and goat over one year period. Lactic acid bacteria were enumerated using 8 semi-selective media, and systematically screened for their antifungal activity against 4 spoilage fungi commonly encountered in dairy products. Depending on the selective medium, between 0.05% (Elliker agar) and 5.5% (LAMVAB agar) screened colonies showed an antifungal activity. The great majority of these active colonies originated from cow (49%) and goat (43%) milks, whereas only 8% were isolated from ewe milk. Penicillium expansum was the most frequently inhibited fungus with 48.5% of colonies active against P. expansum among the 1235 isolated, followed by Mucor plumbeus with 30.6% of active colonies, Kluyveromyces lactis with only 12.1% of active colonies and Pichia anomala with 8.7% of active colonies. In the tested conditions, 94% of the sequenced active colonies belonged to Lactobacillus. Among them, targeted fungal species differed according to the Lactobacillus group, whose presence largely depended on year period and milk origin. The Lb. casei and Lb. reuteri groups, predominantly recovered in summer/fall, were overrepresented in the population targeting M. plumbeus, whereas isolates from the Lb. plantarum group, predominantly recovered in spring, were overrepresented in the population targeting K. lactis, the ones belonging to the Lb. buchneri group, predominantly recovered in spring, were overrepresented in the population targeting P. anomala. Raw milk, especially cow and goat milks from the summer/fall period appeared to be a productive reservoir for antifungal lactobacilli.

Quantification of Penicillium camemberti and P. roqueforti mycelium by real-time PCR to assess their growth dynamics during ripening cheese.

Real-time PCR has been applied to quantify mycelium of Penicillium camemberti and Penicillium roqueforti during ripening of model cheese curd and surface mould-ripened cheeses. Total fungal DNA was first validated as an indicator of mycelial biomass in pure liquid culture and then in model curds at different stages of ripening. To imitate cheese matrix effects, DNA was extracted from curd mixed with known amounts of fresh mycelium of P. camemberti or P. roqueforti and was used as biomass standards for further quantitative real-time PCR. Mycelial mass per cheese (mg/g) was then directly obtained from fluorescence data. In model cheese curd, mycelial mass of P. camemberti increased from 2.8 at d4 to 596 mg/g at d11 whereas P. roqueforti increased from 0.3 to 6.3 mg/g during the same period. P. camemberti showed a fast development in Coulommiers from d2 to d9 (66 to 119 mg/g) and a 100-fold increase in Carré (0.85 to 85 mg/g). While mycelial biomass reached a maximum at d9 in Coulommiers, it still developed in Carré until d45. For the first time, cheese manufacturers have a powerful technique to monitor mycelial growth dynamics of their fungal cultures, which represents an important step for controlling cheese making.

Assessment of the microbial diversity at the surface of Livarot cheese using culture-dependent and independent approaches.

The microbial diversity of the surface of a commercial red-smear cheese, Livarot cheese, sold on the retail market was studied using culture-dependent and independent approaches. Forty yeasts and 40 bacteria from the cheese surface were collected, dereplicated using single-strand conformation polymorphism (SSCP) analysis and identified using rRNA gene sequencing for the culture-dependent approach. The culture-independent approach involved cloning and sequencing of the 16S rRNA gene and SSCP analysis from total DNA extracted from the cheese. The most dominant bacteria were Microbacterium gubbeenense, Leucobacter komagatae and Gram-negative bacteria from the Gamma-Proteobacteria class. Fluorescence in situ hybridization (FISH) analysis was also used to study the cheese microbial diversity with class-level and specific rRNA-targeted probes for bacteria and yeasts, respectively. FISH analysis confirmed that Gamma-Proteobacteria were important microorganisms in this cheese. Four specific FISH probes targeting the dominant yeasts present in the cheese, Candida catenulata, Candida intermedia, Geotrichum spp. and Yarrowia lipolytica, were also designed and evaluated. These probes allowed the detection of these yeasts directly in cheese. The use of the rRNA gene-based approach combined with FISH analysis was useful to investigate the diversity of a surface microbial consortium from cheese.

Survival of surface ripening cultures during storage and monitoring their development on cheese.

AIMS: To study the survival of bacteria isolated from the surface of smear cheese and monitor their development during cheese ripening. METHODS AND RESULTS: The storage of five potential bacterial surface-ripening cheese cultures, Brevibacterium aurantiacum, Corynebacterium casei, Corynebacterium variable, Microbacterium gubbeenense and Staphylococcus saprophyticus, in maximum recovery diluent (MRD), containing 0.85% w/v or 5% w/v NaCl, at 21 or 4 degrees C for 40 days, was investigated. All five strains studied survived well with a maximum decrease of c. 2.5 log(10) CFU ml(-1) after storage for 40 days at 4 degrees C in 0.85% or 5% w/v NaCl. Survival, especially of C. variable, was less at 21 degrees C. The development of defined ripening cultures containing C. casei and Debaryomyces hansenii on two farmhouse cheeses was also evaluated. Using pulsed-field gel electrophoresis (PFGE) for the bacteria and mitochondrial DNA restriction fragment length polymorphism (mtDNA-RFLP) for the yeast, it was shown that the ripening cultures could be re-isolated in high numbers, 10(8) CFU cm(-2) for C. casei and 10(6) CFU cm(-2) for D. hansenii, from the cheese surface after 2.5 weeks of ripening. CONCLUSIONS: Ripening strains of surface ripening cultures can be stored in MRD containing 5% w/v salt at 4 degrees C for at least 40 days. Such cultures are recovered in high numbers from the cheese during ripening. SIGNIFICANCE AND IMPACT OF STUDY: This study has provided a low-cost and efficient way to store bacteria that could be used as ripening cultures for smear cheese. Such cultures can be recovered in high numbers from the cheese surface during ripening.

Sources of the adventitious microflora of a smear-ripened cheese.

AIMS: To determine the relationships between the major organisms from the cheese-making personnel and environment and the surface of a smear cheese. METHODS AND RESULTS: 360 yeast and 593 bacteria from the cheese surface, the dairy environment and the hands and arms of personnel were collected. Pulsed-field gel electrophoresis, repetitive sequence-based polymerase chain reaction and 16S rDNA sequencing were used for typing and identifying the bacteria, and mitochondrial DNA restriction fragment length polymorphism and Fourier-transform infrared spectroscopy for typing and identifying the yeast. The three most dominant bacteria were Corynebacterium casei, Corynebacterium variabile and Staphylococcus saprophyticus, which were divided into three, five and seven clusters, respectively, by macrorestriction analysis. The same clones from these organisms were isolated on the cheese surface, the dairy environment and the skin of the cheese personnel. Debaryomyces hansenii was the most dominant yeast. CONCLUSIONS: A 'house' microflora exists in the cheese plant. Although the original source of the micro-organisms was not identified, the brines were an important source of S. saprophyticus and D. hansenii and, additionally, the arms and hands of the workers the sources of C. casei and C. variabile. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time that the major contribution of the house microflora to the ripening of a smear-ripened cheese has been demonstrated.

Metabolic events mediating early killing of host cells infected by Shigella flexneri.

J774, a continuous macrophage cell-line, was infected by M90T, an invasive isolate of Shigella flexneri serotype 5 and BS176, its non invasive derivative--which does not harbor the 220 kbase virulence plasmid pWR100. Killing of host cells by intracellular M90T, commenced one hour after infection and was completed by 4 hours. Intracellular BS176 did not kill cells during the same period. Cell protein biosynthesis was totally inhibited by both strains within 2 hours of infection thus indicating that shiga-like toxin 1 (SLT1) could not account for early killing. On the other hand a sharp decrease in intracellular ATP was observed after 1 hour in cells infected with M90T. No significant increase in ATPase activity could be detected. A sharp increase in pyruvate production starting immediately after infection indicated impairement in mitochondrial respiration, which accounts for most ATP produced intracellularly. In addition, fermentation appeared to be totally blocked thus leaving no chance of the infected cells regenerating NAD. Concurrent increase in cAMP concentration within the first hour of infection may contribute to the rapid and efficient cell killing. Cells infected by BS176 always showed an intermediate phenotype (i.e. ATP depletion, pyruvate increase, lactate decrease). Early lysis of the phagocytic vacuole by M90T may account for this difference by allowing toxic products of the bacteria to diffuse more efficiently within the cytosol.

Movement along actin filaments of the perijunctional area and de novo polymerization of cellular actin are required for Shigella flexneri colonization of epithelial Caco-2 cell monolayers.

Shigella flexneri invades eucaryotic cells and grows in the cytoplasm. Lysis of the phagosomal membrane is a prerequisite for both intracellular multiplication and movement of the bacteria that gain direct access to the host cell actin. In HeLa cells, bacteria generate their own movement essentially by inducing actin polymerization. Polymerization of actin enables them to move rapidly and randomly in the cytoplasm and to spread from one cell to another through protrusions of the host cell membrane. This movement was designated the Ics phenotype. In contrast, in chicken embryo fibroblasts, bacteria move along actin filaments in a very organized manner, following the cytoskeletal architecture; this movement was designated the Olm phenotype. Bacterial movement is a major virulence factor in that it is necessary for efficient colonization of the intestinal epithelium of infected macaque monkeys. Further characterization of the cellular events that lead to colonization of the colonic intestinal epithelium was needed. In order to characterize the movement in vitro in a cell assay system more closely related to the intestinal epithelium, we used human colonic epithelial Caco-2 cells. The movement of bacteria as observed by using immunofluorescence and confocal microscopy appeared to result from the expression of both the Olm and Ics phenotypes. The former allowed colonization of cells along the actin filament ring of the perijunctional area. The latter promoted passage from one cell to adjacent cells. This in vitro pattern of movement and multiplication gives S. flexneri, once it has entered an epithelial cell, the unique capacity to spread through the entire epithelial layer without having further contact with the extracellular compartment.